The present invention relates to a selective bifunctional sequential linker and a method for using it to link two different amine-containing compounds. The method and linker of the invention are especially useful for preparing anitbody conjugates.
It is useful in a variety of situations to link a compound bearing a primary or secondary amine function to another compound bearing an amine function or another function. For example, a solid polymer support is often derivatized to form a pendant amine-containing polymer, after which it is linked to molecules of interest as components of an assay, e.g., an immunoassay. Such a polymer could also function as an immunoadsorbant for affinity chromatography.
Various bifunctional linkers can be used to achieve such linkages. Succinic anhydride converts an amine-functionalized polymer to a carboxyl-functionalized material, which can be reacted with another amine ligand using any of a variety of carboxyl activators, e.g., dicyclohexylcarbodiimide and its analogues. Alkylation of the amine with, e.g., N-succinimidyl bromoacetate (bromoacetic acid ester of N-hydroxysuccinimide) results in another carboxyl derivative which can be reacted with an amine or hydroxyl ligand to form an amide or ester. Acylation of the amine with N-acetylhomocysteine thiollactone (5-acetamido-2-thiacyclopentanone) results in a derivative having a pendant thiol, which can be esterified or otherwise reacted to form an adduct.
Two immunoglobulins can be linked to form a cross-linked reagent useful as a component in an immunoassay, as disclosed in U.S. Pat. No. 4,433,059, the entire disclosure of which is incorporated herein by reference. In this application, one immunoglobulin was reacted at amine functions with an amine-reactive function on the linker, while the other immunoglobulin was reacted at thiol functions generated by cleavage of disulfide bonds or by deacetylation of S-acetylmercaptosuccinyl groups, the thiol function being reacted with, e.g., maleimide, iodoacyl or 2-pyridyldithio functions on the linker.
An amine-containing hapten can be linked to pendant amine groups of lysyl residues on an immunogenic protein to form an immunogen, as disclosed in, e.g., U.S. Pat. No. 4,486,344, which is incorporated herein in its entirety by reference. The linkers disclosed for this purpose were characterized as "phosgene equivalents", and included such reactive compounds as carbonyldiimidazole, disuccinimidyl carbonate, p-nitrophenyl chloroformate and the like. These reagents were generally used to form highly reactive intermediates, typically imidazoylureas, which were reacted in situ to form urea linkages with protein, which in this case was bovine serum albumin. The reference notes that such imidiazoylureas are normally not isolated, and it is doubtful whether it could be readily isolated and stably stored for any substantial period of time.
Diisothiocyanates are known as linkers for amine functions. However, formation of a monofunctional intermediate is complicated by significant addition at both isothiocyanate groups, unless a substantial excess of linker is used, which must then be removed prior to reaction with the second amine ligand.
A need therefore continues to exist for a selective bifunctional sequential linker which improves upon those known to the art.